This invention relates to nucleic acid molecules encoding the phage endolysins: PlyCP10, PlyCP18, PlyCP33, and PlyCP41, PlyCP18 and PlyCP33 harbor L-alanine amidase catalytic domains, and PlyCP10 and PlyCP41 have glycosyl hydrolase catalytic domains which specifically attack the peptidoglycan cell wall of Clostridium perfringens bacteria which contributes to severe gut infections (necrotic enteritis) in animals such as poultry, and new-born cattle and swine.
Clostridium perfringens is a Gram-positive, spore forming, anaerobic bacterium commonly present in the intestines of humans and animals. C. perfringens is classified into one of five types (A, B, C, D, or E) based on the toxin production. Spores of the pathogen can persist in soil, feces or the environment, and the bacterium causes many severe infections of animals and humans. Some strains of C. perfringens type A produce an enterotoxin (CPE) during sporulation that are responsible for food-borne disease in humans (Smedley et al. 2004. Rev. Physiol. Bioch. P. 152:183-204; Sawires and Songer. 2006. Anaerobe 12:23-43; Scallan et al. 2011. Emerg. Infect. Dis. 17:16-22). C. perfringens can cause food poisoning, gas gangrene, necrotic enteritis, and non-foodborne gastrointestinal infections in humans.
Necrotic enteritis is a peracute disease syndrome and is the most common and financially devastating bacterial disease in modern broiler flocks. The clinical form in poultry is caused by alpha toxin-producing C. perfringens type A. Although the clinical illness is usually very short, mortality in an unprotected poultry flock can be devastating. Often the only sign of necrotic enteritis in a flock is a sudden increase in mortality. In addition to increased mortality, necrotic enteritis may present as birds with depression, ruffled feathers, and dark diarrhea. The disease persists in a flock for between about 5-10 days, with mortality between 2-50%. Necrotic enteritis can be controlled by antimicrobial drugs administered at prophylactic doses either in water or in feed; however, there is increasing public opposition to the use of antibiotics in animal feeds.
In the European Union (EU) antimicrobial growth promotants (AGPs) were banned from animal feeds on Jan. 1, 2006 (Regulation 1831/2003/EC) because of concerns about the increasing prevalence of antibiotic resistances among bacteria (Huyghebaert et al. 2011. Vet. J. 187:182-188; Millet and Maertens. 2011. Vet. J. 187:143-144). In 2015, the state of California passed a law banning the routine use of antibiotics in livestock (Retrieved from the Internet: mercurynews.com/california/ci_28951303/antibiotics). Earlier in 2015, McDonald's, the fast-food corporation, announced that it was going to use antibiotic-free chickens (Retrieved from the Internet:nytimes.com/2015/03/05/business). These events are likely precursors to further bans of the use of antibiotics in animal-feed in other states, or even a national ban in the U.S., within the next few years. Without traditional antibiotics for the prevention of necrotic enteritis and other diseases caused by C. perfringens, such diseases could potentially become a far greater problem for the livestock industry. Removal of these antimicrobials will dictate the need for alternative antimicrobials in order to achieve the same high level of food-animal production achieved with AGPs. Also changes within the gastrointestinal microbial flora of food-producing animals will result in the need for a more complete understanding of the gut microbial ecology (Wise & Siragusa. 2007. J Appl. Microbiol. 102:1138-1149; Oakley et al. 2013. Plos One 8(2): e57190) so that appropriate antibiotic alternatives may be developed for use during food-animal production (Seal et al. 2013. Anion. Health Res. Rev. 14:78-87).
Prior to the discovery and widespread use of antibiotics, bacterial infections were treated by administering bacteriophages and were marketed by L'Oreal in France. Although Eli Lilly Co. marketed phage products for human use until the 1940's, early clinical studies with bacteriophages were not extensively undertaken in the United States and Western Europe after that time. Bacteriophages were and continue to be sold in the Russian Federation and Eastern Europe as treatments for bacterial infections (Sulakvelidze et al. 2005. Drug Discovery Today 10:807-809). There has been a resurgent interest in bacteriophage biology and use of phage gene products as antibacterial agents (Liu et al. 2004. Nature Biotech. 22:185-191; Pastagia et al. 2013. J. Med. Microbiol. 62:1506-1516; Schmelcher et al. 2012. Future Microbiol. 7:1147-1171; Rodriguez-Rubio et al. 2014. Crit. Rev. Microbiol. 39:427-434; Seal, B. S. 2013. Poultry Sci. 92:526-533). The potential use of lytic bacteriophages and/or their lytic enzymes has been of considerable interest for veterinary and human medicine, as well as the bioindustry worldwide due to antibiotic resistance issues among bacterial pathogens. Recently, the U.S. Food and Drug Administration approved a mixture of anti-Listeria viruses as a food additive to be used in processing plants for spraying onto ready-to-eat meat and poultry products to protect consumers from Listeria monocytogenes (Bren, L. 2007. FDA Consum. 41:20-22). Although bacteriophages have been considered as potentially important alternatives to antibiotics (Sulakvelidze et al., supra; Lu and Koeris. 2011. Curr. Opin. Microbiol. 14:524-531; Maura and Debarbieux. 2011. Appl. Microbiol. Biotech. 90:851-859), it is important to emphasize that development of bacterial resistances to their viruses occurs. Evolution of phage receptors, super-infection exclusion, restriction enzyme-modification systems and abortive infection systems such as bacterial CRISPR sequences are all mechanisms that bacteriophage hosts utilize to avoid infection (Labrie et al. 2010. Nature Rev. Microbiol. 8:317-327), arguing for use of bacteriophage lytic proteins.
Antibiotic resistance among pathogens is believed to develop, in part, through the use of broad range antibiotics, which affect not only the target pathogen, but can also select for resistance in other bacteria (e.g. commensals). The use of a highly specific antimicrobial would target fewer species, and thus is less likely to contribute to the broad range resistance development now apparent with commonly used broad range antibiotics. Bacteriophage endolysins are uniquely specific to their host (or closely related species); bacteriophage and bacterial hosts have co-evolved. It is difficult to prove that resistance cannot develop to endolysins, but to date, none has been reported and this fact alone makes this product a candidate for addition to the battery of antimicrobials available to both veterinary medicine and the clinician. If resistant strains are not produced, this would be an important antimicrobial for use and efficacy.